亀井 克彦

We constructed deletion mutants of Cryptococcus neoformans var neoformans (serotype D) genes encoding late ergosterol biosynthetic pathway enzymes and found that the mutations enhanced susceptibility to various drugs including micafungin, one of the echinocandins, to which wild-type Cryptococcus strains show no susceptibility. Furthermore, through isolation of a mutant resistant to micafungin from a micafungin-sensitive erg mutant and genetic analysis of it, we found that the responsible mutation occurred in the hotspot 2 of FKS1 encoding beta-1, 3-glucan synthase, indicating that micafungin inhibited the growth of the erg mutant via inhibiting Fks1 activity. Addition of ergosterol to the culture of the erg mutants recovered the resistance to micafungin, suggesting that the presence of ergosterol in membrane inhibits the accession of micafungin to its target. We found that a loss of one of genes encoding subunits of v-ATPase, VPH1, made Cryptococcus cells sensitive to micafungin. Our observation that the erg2 vph1 double mutant was more sensitive to micafungin than either single mutant suggests that these two genes act differently in becoming resistant to micafungin. The erg mutants allowed us to study the physiological significance of beta-1, 3-glucan synthesis in C. neoformans; the inhibition of beta-1, 3-glucan synthesis induced cell death and changes in cellular morphology. By observing the erg mutant cells recovering from the growth inhibition imposed by micafungin, we recognized beta-1, 3-glucan synthesis would suppress filamentous growth in C. neoformans.

Histoplasmosis is a common endemic mycosis that is usually asymptomatic but occasionally results in severe illness. Histoplasmosis and its causative agent, Histoplasma capsulatum, are found worldwide but rarely in Japan. In recent years, however, the number of histoplasmosis patients in Japan has increased. In addition, to our knowledge, there are no previous reports of increased serum soluble interleukin-2 receptor (sIL-2R) levels in patients with histoplasmosis. We report a case series of histoplasmosis in three Japanese temporary workers in Manzanillo, Mexico. All three patients developed a persistent high fever and general fatigue. Laboratory tests showed increased C-reactive protein levels and mild liver dysfunction. All patients also showed increased soluble interleukin-2 receptor (sIL-2R) levels. Chest computed tomography revealed multiple nodules in both lung fields. All patients were positive for serum anti-Histoplasma antibodies, and two patients were positive for Histoplasma on polymerase chain reaction tests. After treatment that included antifungals, their conditions gradually improved and laboratory data normalized. Although one patient developed respiratory failure, this patient recovered with antifungal therapy in combination with methylprednisolone. Serum sIL-2R levels in all patients gradually declined to normal levels, indicating their recovery from Histoplasma infection. From our experience with these patients, sIL-2R levels may be a useful biomarker for patients with histoplasmosis.

Background: Some patients with severe asthma also have fungal sensitization and are considered to have severe asthma with fungal sensitization. However, there is limited information on the clinical features of SAFS. Objective: To investigate the clinical characteristics of severe asthma with fungal sensitization. Methods: The present study enrolled 124 patients with severe asthma. We evaluated clinical aspects, such as various serum cytokines, fractional exhaled nitric oxide, pulmonary function, and serum immunoglobulin E (IgE). Fungal sensitization was assessed by determining serum levels of IgE specific to fungal allergens (Aspergillus, Alternaria, Candida, Cladosporium, Penicillium, and Trichophyton species and Schizophyllumcommune). The protocol was registered at a clinical trial registry (www.umin.ac.jp/ctr/index-j.htm; UMIN 000002980). Results: Thirty-six patients (29%) showed sensitization to at least 1 fungal allergen. The most common species were Candida (16%), Aspergillus (11%), and Trichophyton (11%). The rate of early-onset asthma (<16 years of age) was higher in patients with fungal sensitization than in those without fungal sensitization (45% vs 25%; P = .02). Interleukin-33 levels were higher in patients with fungal sensitization than in those without fungal sensitization. Of patientswith atopic asthma, Asthma Control Test scores were worse in patients with multiple fungal sensitizations than in patients with a single fungal sensitization or those without fungal sensitization. Conclusion: Severe asthma with fungal sensitization is characterized by early onset of disease and high serum levels of interleukin-33. Multiple fungal sensitizations are associated with poor asthma control. (C) 2017 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Recently, azole-resistant Aspergillus fumigatus containing a 34-bp or 46-bp tandem repeat in the promoter region of cyp51A combined with amino acid substitution(s) has appeared in the environment worldwide, including several Asian countries. In this study, we isolated the 34-bp tandem repeat-containing azole-resistant A. fumigatus strain OKH50 from a patient in Japan in May 2016. The patient had not been treated with medical azoles before the strain isolation, suggesting that the resistant property was acquired before infection. In addition, the patient had not traveled overseas. Our analysis of short tandem repeats of the strain indicates that the strain is strongly related to the 34-bp tandem repeat-containing isolates from European countries and Asia-Oceania countries but not to susceptible isolates from Japan, suggesting that the strain was introduced from overseas and might spread in Japan. (C) 2016 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

It is well known that 5-fluoroorotic acid (5-FOA)-resistant mutants isolated from wild-type Cryptococcus neoformans are exclusively either ura3 or ura5 mutants. Unexpectedly, many of the 5-FOA-resistant mutants isolated in our selective regime were Ura(+). We identified CNM00460 as the gene responsible for these mutations. Cnm00460 belongs to the nucleobase cation symporter 1/purine-related transporter (NCS1/PRT) super family of fungal transporters, representative members of which are uracil transporter, uridine transporter and allantoin transporter of Saccharomyces cerevisiae. Since the CNM00460 gene turned out to be involved in utilization of orotic acid, most probably as transporter, we designated this gene Orotic Acid Transporter 1 (OAT1). This is the first report of orotic acid transporter in this family. C. neoformans has four members of the NCS1/PRT family, including Cnm00460, Cnm02550, Cnj00690, and Cnn02280. Since the cnm02550a Delta strain showed resistance to 5-fluorouridine, we concluded that CNM02550 encodes uridine permease and designated it URidine Permease 1 (URP1). We found that oat1 mutants were sensitive to 5-FOA in the medium containing proline as nitrogen source. A mutation in the GAT1 gene, a positive transcriptional regulator of genes under the control of nitrogen metabolite repression, in the genetic background of oat1 conferred the phenotype of weak resistance to 5-FOA even in the medium using proline as nitrogen source. Thus, we proposed the existence of another orotic acid utilization system (tentatively designated OAT2) whose expression is under the control of nitrogen metabolite repression at least in part. We found that the OAT1 gene is necessary for full pathogenic activity of C. neoformans var. neoformans.

The performance of three molecular biology techniques, i.e., DNA microarray, loop-mediated isothermal amplification (LAMP), and real-time PCR were compared with DNA sequencing for properly identification of 20 isolates of Fusarium spp. obtained from blood stream as etiologic agent of invasive infections in patients with hematologic malignancies. DNA microarray, LAMP and real-time PCR identified 16 (80%) out of 20 samples as Fusarium solani species complex (FSSC) and four (20%) as Fusarium spp. The agreement among the techniques was 100%. LAMP exhibited 100% specificity, while DNA microarray, LAMP and real-time PCR showed 100% sensitivity. The three techniques had 100% agreement with DNA sequencing. Sixteen isolates were identified as FSSC by sequencing, being five Fusarium keratoplasticum, nine Fusarium petroliphilum and two Fusarium solani. On the other hand, sequencing identified four isolates as Fusarium non-solani species complex (FNSSC), being three isolates as Fusarium napiforme and one isolate as Fusarium oxysporum. Finally, LAMP proved to be faster and more accessible than DNA microarray and real-time PCR, since it does not require a thermocycler. Therefore, LAMP signalizes as emerging and promising methodology to be used in routine identification of Fusarium spp. among cases of invasive fungal infections.

Aspergillus niger and its related species, known as Aspergillus section Nigri, are ubiquitously distributed across the globe and are often isolated from clinical specimens. In Japan, Aspergillus section Nigri is second most often isolated from clinical specimens following Aspergillus fumigatus. We determined the species of Aspergillus section Nigri isolated in Japan by DNA sequencing of partial beta-tubulin genes and investigated drug susceptibility by the CLSI M38-A2 method. The collection contained 20 Aspergillus niger, 59 Aspergillus welwitschiae, and 39 Aspergillus tubingensis strains. Drug susceptibility testing revealed 30 to 55% of A. niger, 6.8 to 18.6% of A. welwitschiae, and 79.5 to 89.7% of A. tubingensis isolates to be less susceptible (so-called resistant) to itraconazole (ITC) and/or voriconazole (VRC) according to the epidemiologic cutoff values (ECVs) proposed for A. niger previously. MIC distributions of ITC or VRC showed no remarkable differences between clinical and environmental isolates. When the cyp51A sequences were compared between susceptible and resistant strains, 18 amino acid mutations were specific for resistant isolates of A. niger and A. tubingensis; however, none of them were confirmed to be associated with azole resistance. Three nonrelated A. welwitschiae isolates possessed a partial deletion in cyp51A, likely attributable to being more susceptible to azoles than other isolates. One of five ITC-resistant A. tubingensis isolates showed higher expression of cyp51A than did susceptible strains. Our results show that cyp51A point mutations may have no association with azole resistance but that in some cases the overexpression of cyp51A may lead to the azole resistance in these species.

Asexual spores (conidia) are reproductive structures that play a crucial role in fungal distribution and survival. As fungal conidia are, in most cases, etiological agents of plant diseases and fungal lung disease, their stress resistance and interaction with their hosts have drawn increasing attention. In the present study, we investigated whether environmental temperature during conidiation affects the stress tolerance of the conidia of the human pathogenic fungus Aspergillus fumigatus. Conidia from a 25 degrees C culture showed a lower tolerance to heat (60 degrees C) and oxidative (H2O2) stresses and a marked resistance to ultraviolet radiation exposure, compared with those produced at 37 and 45 degrees C. The accumulation of trehalose was lower in the conidia from the 25 degrees C culture. Furthermore, the conidia from the 25 degrees C culture showed darker pigmentation and increased transcripts of dihydroxynaphthalene (DHN)melanin biosynthesis-related genes (i.e., pksP, arp1, and arp2). An RNA-sequencing analysis revealed that the transcription level of the trypacidin (tpc) gene cluster, which contains 13 genes, was sharply and coordinately activated in the conidia from the 25 degrees C culture. Accordingly, trypacidin was abundant in the conidia from the 25 degrees C culture, whereas there was little trypacidin in the conidia from the 37 degrees C culture. Taken together, these data show that the environmental temperature during conidiation affects conidial properties such as stress tolerance, pigmentation, and mycotoxin accumulation. To enhance our knowledge, we further explored the temperature-dependent production of DHN-melanin and trypacidin in clinical A. fumigatus isolates. Some of the isolates showed temperature-independent production of DHN-melanin and/or trypacidin, indicating that the conidia-associated secondary metabolisms differed among the isolates.

Azole-resistant strains of Aspergillus fumigatus containing a tandem repeat in the cyp51A promoter and amino acid substitution(s) have been isolated in the environment worldwide; however, this type of resistant strain had never been isolated from the environment in Japan. Our previous study indicated that an azole-resistant A. fumigatus strain OKH50 containing a 34-bp tandem repeat in cyp51A promoter with L98H substitution in Cyp51A was isolated from a patient in Obihiro of Hokkaido, Japan. In this study, we collected azole-resistant Aspergillus spp. by air sampling from the environment in Japan. One Aspergillus-like colony was isolated from one of 10 sampling sites surveyed. The strain Env1 was confirmed as A. fumigatus by nucleotide sequencing and possessed a 34-bp tandem repeat in the promoter region of cyp51A with L98H substitution in Cyp51A. A. fumigatus Env1 has the identical short tandem repeat pattern with the OKH50 strain, indicating that these strains are closely related with each other. Additionally, the short tandem repeat pattern is closely related to Danish and Iranian environmental isolates, suggesting that azole-resistant strains have crossed transnational boundaries and are now present in Japan, and therefore, further analysis throughout Japan is required to determine the distribution of this type of azole-resistant A. fumigatus.

AFRS caused by S. commune diagnosed using specific antibodies and genetic identification. Background: Schizophyllum commune is a widespread species of fungus that grows on a variety of trees and decaying wood. It only rarely causes human disease such as allergic bronchopulmonary mycosis, fungal rhinosinusitis and allergic fungal rhinosinusitis (AFRS). AFRS caused by S. commune is the most unusual among these diseases. Case reports: we present two cases of S. commune-induced AFRS in a 34yearold and a 27-year-old woman diagnosed by elevated IgE antibodies, computed tomography findings and histopathological examinations of mucous and culture. Conclusion: the two patients showed positive specific IgE and IgG antibodies against S. commune using an enzymelinked immunosorbent assay. We confirmed a decrease in the values of specific antibodies after remission in a patient.

Successful treatment of aspergillosis caused by Aspergillus fumigatus is threatened by an increasing incidence of drug resistance. This situation is further complicated by the finding that strains resistant to azoles, the major antifungal drugs for aspergillosis, have been widely disseminated across the globe. To elucidate mechanisms underlying azole resistance, we identified a novel transcription factor that is required for normal azole resistance in Aspergill-us fungi including A. fumigatus, Aspergillus oryzae, and Aspergillus nidulans. This fungal-specific Zn-2-Cys(6) type transcription factor AtrR was found to regulate expression of the genes related to ergosterol biosynthesis, including cyp51A that encodes a target protein of azoles. The atrR deletion mutant showed impaired growth under hypoxic conditions and attenuation of virulence in murine infection model for aspergillosis. These results were simi-lar to the phenotypes for a mutant strain lacking SrbA that is also a direct regulator for the cyp51A gene. Notably, AtrR was responsible for the expression of cdr1B that encodes an ABC transporter related to azole resistance, whereas SrbA was not involved in the regula-tion. Chromatin immunoprecipitation assays indicated that AtrR directly bound both the cyp51A and cdr1B promoters. In the clinically isolated itraconazole resistant strain that har-bors a mutant Cyp51A (G54E), deletion of the atrR gene resulted in a hypersensitivity to the azole drugs. Together, our results revealed that AtrR plays a pivotal role in a novel azole resistance mechanism by co-regulating the drug target (Cyp51A) and putative drug efflux pump (Cdr1B).

Organizing pneumonia (OP) is a nonspecific response to various forms of lung injury and has been reported in association with several infectious agents. However, little is known about the relationship between OP and chronic pulmonary aspergillosis, and the mechanism of this linkage has not been elucidated. Here, we present a case of chronic pulmonary aspergillosis that led to the development of OP, which was successfully treated with corticosteroid and surgical intervention. In a review of the literature, we aim to highlight the possible relationship between OP and chronic pulmonary aspergillosis.

In Japan, Fusarium species are known etiological agents of human fungal infection however, there has been no report of a large-scale epidemiological study on the etiological agents of fusariosis. A total of 73 Fusarium isolates from patients with invasive fusariosis (IF, n = 36) or superficial fusariosis (SF, n = 37), which were obtained at hospitals located in 28 prefectures in Japan between 1998 and 2015, were used for this study. Fusarium isolates were identified using Fusarium- and Fusarium solani species complex (FSSC)-specific real-time PCR and partial DNA sequences of the elongation factor-1 alpha (EF-1α) gene and the nuclear ribosomal internal transcribed spacer (ITS)region. FSSC was predominately isolated from both patients with IF and SF (IF, 77.8% and SF, 67.6%). Distribution of the phylogenetic species of FSSC isolates from patients with IF and SF exhibited different spectra specifically, F. keratoplasticum (FSSC 2) (25.0%)was the most frequent isolate from patients with IF, whereas F. falciforme (FSSC 3+ 4) (32.4%)was the most frequent isolate from patients with SF. Fusarium sp. (FSSC 5)was the second most frequent isolate from both patients with IF and SF (IF, 22.2% and SF, 24.3%). Notably, F. petroliphilum (FSSC 1)was isolated only from patients with IF. Each species was isolated from a broad geographic area, and an epidemic was not observed. This is the first epidemiological study of Fusarium species causing IF and SF in Japan.

76歳男。陰嚢部の硬結を主訴とした。急性骨髄性白血病治療中の患者で陰嚢の硬結と疼痛にて紹介受診となり、ヘルペス感染症を考えて抗ウイルス薬を投与するも陰嚢前面、陰茎に黒色壊死・痂皮を伴う皮膚潰瘍が拡大し、新たに右眉毛外側に黒色壊死を伴う硬結(20mm大)が出現した。陰嚢の硬結の皮膚生検では真皮に著明な炎症細胞浸潤と壊死、中央部の潰瘍底に多数の真菌の菌体を認め、KOH鏡検で菌糸陽性、培養では白色調で表面綿毛状の集落形成が確認された。また、スライドカルチャーでは三日月型の大分生子を認め、遺伝子解析によりFusarium solani species complexと同定された。ボリコナゾールの全身投与により皮膚症状の進行は抑制されたが、原疾患の増悪により死亡した。

<p>12 歳の男児，サッカー部に所属している。2015 年 5 月より左膝に瘙痒を伴う紅斑が出現し，近医で苛性カリ鏡検法 (KOH 検査) にて陰性であり，ステロイド外用加療で改善がなく，同年 6 月に当科を受診した。左膝に 6 × 5 cm の角化性紅斑がみられ，再度表面の鱗屑で KOH 検査を施行したが陰性であった。 しかし毛包を含む皮膚組織での真菌培養検査にて真菌の発育がみられ，同菌株を用いた遺伝子解析で DNA 塩基配列が標準 <i>Trichophyton tonsurans</i>(<i>T. tonsurans</i>) 株と 100％一致し，<i>T. tonsurans</i> による体部白癬と確定診断した。<i>T. tonsurans</i> による体部白癬は，これまでにも報告はあるが稀であり，その臨床像は多彩である。確定診断には組織培養を含めた徹底した精査が必要であると考えた。</p>

Talaromyces marneffei is a dimorphic fungus endemic mainly in southeast and south Asia. It causes severe mycosis, usually in immunocompromised individuals, such as those with human immunodeficiency virus (HIV) infection. Concomitant infection with T. marneffei and other opportunistic pathogens is plausible because the majority of T. marneffei infections occur in patients with advanced HIV infection. Nonetheless, coinfection in the same site has rarely been reported, and poses a considerable diagnostic and therapeutic challenge. We report the case of an HIV-infected Japanese patient who had lived in Thailand for 6 years. The patient developed T. marneffei and Mycobacterium tuberculosis coinfection, and both pathogens were isolated from the same sites: a blood specimen and a lymph node aspirate. Clinicians should be aware of concomitant infection with T. marneffei and other pathogens in patients with advanced HIV disease who are living in or who have visited endemic areas.

Background: Fungal conidia are usually dormant unless the extracellular conditions are right for germination. Despite the importance of dormancy, little is known about the molecular mechanism underlying entry to, maintenance of, and exit from dormancy. To gain comprehensive and inter-species insights, transcriptome analyses were conducted across Aspergillus fumigatus, Aspergillus niger, and Aspergillus oryzae. Results: We found transcripts of 687, 694, and 812 genes were enriched in the resting conidia compared with hyphae in A. fumigatus, A. niger, and A. oryzae, respectively (conidia-associated genes). Similarly, transcripts of 766, 1,241, and 749 genes were increased in the 1 h-cultured conidia compared with the resting conidia (germination-associated genes). Among the three Aspergillus species, we identified orthologous 6,172 genes, 91 and 391 of which are common conidia- and germination-associated genes, respectively. A variety of stress-related genes, including the catalase genes, were found in the common conidia-associated gene set, and ribosome-related genes were significantly enriched among the germination-associated genes. Among the germination-associated genes, we found that calA-family genes encoding a thaumatin-like protein were extraordinary expressed in early germination stage in all Aspergillus species tested here. In A. fumigatus 63 % of the common conidia-associated genes were expressed in a bZIP-type transcriptional regulator AtfA-dependent manner, indicating that AtfA plays a pivotal role in the maintenance of resting conidial physiology. Unexpectedly, the precocious expression of the germination-associated calA and an abnormal metabolic activity were detected in the resting conidia of the atfA mutant, suggesting that AtfA was involved in the retention of conidial dormancy. Conclusions: A comparison among transcriptomes of hyphae, resting conidia, and 1 h-grown conidia in the three Aspergillus species revealed likely common factors involved in conidial dormancy. AtfA positively regulates conidial stress-related genes and negatively mediates the gene expressions related to germination, suggesting a major role for AtfA in Aspergillus conidial dormancy.

Azoles are widely used for controlling fungal growth in both agricultural and medical settings. The target protein of azoles is CYP51, a lanosterol 14-alpha-demethylase involved in the biosynthesis of ergosterol. Recently, a novel azole resistance mechanism has arisen in pathogenic fungal species Aspergillus fumigatus. Resistant strains contain a 34-bp or 46-bp tandem repeat (TR) in the promoter of cyp51A, and have disseminated globally in a short period of time. In this study, we investigated whether an azole-resistant strain with a 46-bp TR (TR46/Y121F/T289A) could be sensitised to azoles by deletion of srbA, encoding a direct regulator of cyp51A. The loss of SrbA did not affect colony growth or conidia production, but decreased expression of cyp51A. The srbA deletion strain showed hypersusceptibility to medical azoles as well as azole fungicides, while its sensitivity to non-azole fungicides was unchanged. This is the first demonstration that deletion of a regulator of cyp51A can sensitise an azole-resistant A. fumigatus strain. This finding may assist in the development of new drugs to help combat life-threatening azole-resistant fungal pathogens.

Invasive aspergillosis is a life-threatening mycosis caused by the pathogenic fungus Aspergillus. The predominant causal species is Aspergillus fumigatus, and azole drugs are the treatment of choice. Azole drugs approved for clinical use include itraconazole, voriconazole, posaconazole, and the recently added isavuconazole. However, epidemiological research has indicated that the prevalence of azole-resistant A. fumigatus isolates has increased significantly over the last decade. What is worse is that azole-resistant strains are likely to have emerged not only in response to long-term drug treatment but also because of exposure to azole fungicides in the environment. Resistance mechanisms include amino acid substitutions in the target Cyp51A protein, tandem repeat sequence insertions at the cyp51A promoter, and overexpression of the ABC transporter Cdr1B. Environmental azole-resistant strains harboring the association of a tandem repeat sequence and punctual mutation of the Cyp51A gene (TR34/L98H and TR46/Y121F/1-289A) have become widely disseminated across the world within a short time period. The epidemiological data also suggests that the number of Aspergillus spp. other than A. fumigatus isolated has risen. Some non-fumigatus species intrinsically show low susceptibility to azole drugs, imposing the need for accurate identification, and drug susceptibility testing in most clinical cases. Currently, our knowledge of azole resistance mechanisms in non-fumigatus Aspergillus species such as A. flavus, A. niger, A. tubingensis, A. terreus, A. fischeri, A. lentulus, A. udagawae, and A. calidoustus is limited. In this review, we present recent advances in our understanding of azole resistance mechanisms particularly in A. fumigatus. We then provide an overview of the genome sequences of non-fumigatus species, focusing on the proteins related to azole resistance mechanisms.

The second cause of death among systemic mycoses, cryptococcosis treatment represents a challenge since that 5-flucytosine is not currently available in Brazil. Looking for alternatives, this study evaluated antifungal agents, alone and combined, correlating susceptibility to genotypes. Eighty Cryptococcus clinical isolates were genotyped by URA5 gene restriction fragment length polymorphism. Antifungal susceptibility was assessed following CLSI-M27A3 for amphotericin (AMB), 5-flucytosine (5FC), fluconazole (FCZ), voriconazole (VRZ), itraconazole (ITZ) and terbinafine (TRB). Drug interaction chequerboard assay evaluated: AMB + 5FC, AMB + FCZ, AMB + TRB and FCZ + TRB. Molecular typing divided isolates into 14 C. deuterogattii (VGII) and C. neoformans isolates were found to belong to genotype VNI (n = 62) and VNII (n = 4). C. neoformans VNII was significantly less susceptible than VNI (P = 0.0407) to AMB; C. deuterogattii was significantly less susceptible than VNI and VNII to VRZ (P < 0.0001). C. deuterogattii was less susceptible than C. neoformans VNI for FCZ (P = 0.0170), ITZ (P < 0.0001) and TRB (P = 0.0090). The combination FCZ + TRB showed 95.16% of synergistic effect against C. neoformans genotype VNI isolates and all combinations showed 100% of synergism against genotype VNII isolates, suggesting the relevance of cryptococcal genotyping as it is widely known that the various genotypes (now species) have significant impact in antifungal susceptibilities and clinical outcome. In difficult-to-treat cryptococcosis, terbinafine and different antifungal combinations might be alternatives to 5FC.

Disseminated infection by Hormographiella aspergillata is extremely rare and small intestine involvement has not been reported previously. A 51-year-old man with myelodysplastic syndrome developed pneumonia after cord blood cell transplantation. Fungal growth from the biopsied lung was identified as H. aspergillata by morphology and the gene analysis. Although antifungal agents including voriconazole and liposomal amphotericin B were administered, he died of disseminated H. aspergillata infection. We review the literature and discuss the treatment and prognosis.

Multi-azole resistant Aspergillus fumigatus carrying TR46/Y121F/T289A was isolated from a patient in Japan in Dec 2013. This strain grouped into the same Glade of the ones which were clinically isolated in France and Germany. A. fumigatus harboring this mutation could be rapidly diffused outside the Eurasian continent. (C) 2016 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

77歳男。右手指背に生じた難治性皮膚潰瘍を主訴とした。既往歴に2型糖尿病、高血圧、高脂血症、狭心症、不整脈、前立腺肥大があった。3ヵ月前に趣味のガーデニング・農作業中に右環指に棘が刺さり、市販の外用薬で治癒しなかった。右環指背に10mm大の辺縁隆起性の紅色結節があり、中央が潰瘍化していた。皮膚生検で慢性肉芽腫性炎症を認め、組織培養でSporothrix schenckii species complexが同定され、皮膚固定型スポロトリコーシスと診断した。尚、遺伝子解析により原因菌種はSporothrix globosaであることが判明した。抗真菌薬の投与を検討したが、循環器科の内服薬に併用禁忌薬のベプリジル塩酸塩水和物があること、肝機能障害があることから、ヨウ化カリウム0.4g/日の内服を開始した。その後、漸増し1.0g/日に達したところで維持量とした。内服開始から数えて計8週間内服したところ、臨床上略治となった。

We developed new cycling probe-based real-time PCR and nested real-time PCR assays for the detection of Histoplasma capsulatum that were designed to detect the gene encoding N-acetylated α-linked acidic dipeptidase (NAALADase), which we previously identified as an H. capsulatum antigen reacting with sera from patients with histoplasmosis. Both assays specifically detected the DNAs of all H. capsulatum strains but not those of other fungi or human DNA. The limited of detection (LOD) of the real-time PCR assay was 10 DNA copies when using 10-fold serial dilutions of the standard plasmid DNA and 50 DNA copies when using human serum spiked with standard plasmid DNA. The nested real-time PCR improved the LOD to 5 DNA copies when using human serum spiked with standard plasmid DNA, which represents a 10-fold higher than that observed with the real-time PCR assay. To assess the ability of the two assays to diagnose histoplasmosis, we analyzed a small number of clinical specimens collected from five patients with histoplasmosis, such as sera (n = 4), formalin-fixed paraffin-embedded (FFPE) tissue (n = 4), and bronchoalveolar lavage fluid (BALF) (n = 1). Although clinical sensitivity of the real-time PCR assay was insufficiently sensitive (33%), the nested real-time PCR assay increased the clinical sensitivity (77%), suggesting it has a potential to be a useful method for detecting H. capsulatum DNA in clinical specimens.

A 16-year-old boy with chronic granulomatous disease presented with pneumonia and rib osteomyelitis. Emericella nidulans var. echinulata was isolated from his sputum. After starting voriconazole, Rasamsonia piperina was isolated from the rib swelling. A combination therapy of voriconazole and micafungin effectively eradicated this invasive mixed-mold infection. In immunocompromised patients, a precise pathogenic diagnosis is clinically useful for administration of an appropriate treatment regimen.

Deoxynivalenol (DON) is an important Fusarium toxin of concern for food safety. The inhalation of powder contaminated with DON is possible and may cause lung toxicity. In this study, we analyzed the gene expression profile of A549 cells treated for 24 hr with 0.2 mu g/mL DON by microarray analysis. In total, 16 genes and 5 noncoding RNAs were significantly affected by DON treatment. The repression of B3GALT4, MEIS3, AK7, SEMA3A, KCNMB4, and SCARA5 was confirmed by quantitative PCR. We investigated the DON toxicity on A549 cells that exogenously expressed these 6 genes. The result indicated that A549 cells that transiently expressed MEIS3 were tolerant to the deleterious effects of DON. Our data show that DON affected the expression of genes with various functions, and suggest that the repression of MEIS3 plays roles in the deleterious effect of DON in A549 lung epithelial cells.

We report herein on the case of a 33-year-old Japanese man in whom an abnormal shadow was detected on chest radiography during a medical checkup after a 1-year-stay in Mexico. Chest computed tomography showed a nodule in the left lower lobe adjacent to the visceral pleura. Histopathologic examination of a thoracoscopic partial pulmonary resection specimen showed coagulation necrosis with a number of yeast-like forms on Grocott staining. In addition, serum anti-&lt;i&gt;Histoplasma &lt;/i&gt;antibody positivity was detected with an enzyme-linked immunosorbent assay, and &lt;i&gt;Histoplasma&lt;/i&gt;-specific nested real-time polymerase chain reaction results were positive in the pulmonary region. Finally, pulmonary histoplasmosis was diagnosed, and treatment with itraconazole was initiated. The patientʼs wife who had accompanied him to Mexico was asymptomatic and was not found to have histoplasmosis based on diagnostic imaging and serological findings. Although rare in Japan, histoplasmosis should be considered in the differential diagnosis of pulmonary lesions in patients who have returned from travel to endemic areas.

&lt;p&gt;65 歳，男性。64 歳時発症の尋常性天疱瘡に対して，プレドニゾロン 13 mg/day およびシクロスポリン130 mg/day を内服中であり，ステロイド糖尿病を合併していた。また，繰り返すサイトメガロウイルス抗原血症にて当科で入退院を繰り返していた。2014 年に同症治療目的で当科に入院中，左大腿伸側に 2×1.5 cm の紅褐色結節を認め，圧迫にて少量の排膿を伴った。病理組織学的には，真皮全層に組織球や好中球を主体とする細胞浸潤を認め，PAS 染色陽性の菌要素が多数みられた。生検組織および膿汁からの分離培養では，いずれもポテトデキストロース寒天培地で，表面が羊毛状，緑褐色で，裏面が褐色から黒色の集落を形成した。スライド培養では洋梨型で，特有な隔壁で縦横に仕切られた分生子を連鎖して認めた。遺伝子解析にて &lt;i&gt;Alternaria alternata&lt;/i&gt; と同定し，深在性皮膚アルテルナリア症と診断した。プレドニゾロンおよびシクロスポリンの漸減を行いつつ，ボリコナゾール 300 mg/day の内服で治療を開始したが，肝機能障害が出現し，イトラコナゾール 200 mg/day の内服に変更した。その後，約 3 週間の経過で皮疹はほぼ平坦化した。深在性皮膚アルテルナリア症は，本邦での報告は自験例を含め 27 例と稀であり，本稿では，2000 年以降の報告例13例についてまとめた。その多くでイトラコナゾールが奏効していた。&lt;/p&gt;

A 39-year-old man presented to our hospital with a four-week history of headache and a two-week history of low-grade fever. Chest X-rays showed a tumor of approximately 50 mm in size in the right lower field. A histopathological examination of a transbronchial lung biopsy specimen from the right S9/10 revealed numerous fungal elements that appeared as encapsulated yeast with clear halos. Gadolinium-enhanced brain magnetic resonance images showed multiple cerebral nodules. Cryptococcus gattii (Genotype VGIIa) was isolated from the bronchial lavage and cerebrospinal fluid specimens. The patient was an immunocompetent Japanese man who had not recently traveled to a C. gattii-endemic area.

Aspergillus lentulus, a sibling species of Aspergillus fumigatus, has been reported as a causative agent of aspergillosis, and exhibited low susceptibility to azole. Here, we present the draft genome sequence of A. lentulus strain IFM 54703T for the first time.

急性骨髄性白血病の地固め療法中に難治性多発肝膿瘍を合併した67歳女性の一例を経験した。吸引生検で糸状菌感染と考えられたが膿瘍・血液ともに培養は陰性であった。当初ミカファンギン、つづいてフルコナゾールを投与したがいずれも無効であった。リポゾーム化アムホテリシンBに変更したところ、徐々に状態は改善した。しかし、治療終了後3ヵ月で再燃、抗真菌剤の肝内濃度を高めるために肝動脈内にカテーテルを留置し、持続動注を行ったところ症状は改善した。7週後にカテーテルが閉塞した後、急激に状態が悪化した。死後、起因菌はCandida tropicalisと判明した。肝動注療法は重症あるいは難治性肝膿瘍の治療の一つになり得ると考えられた。(著者抄録)

We investigated the inhibitory effects of antibacterial, biocidal, and antifungal agents against Fusarium spp. Seven Fusarium spp: four F. falciforme (Fusarium solani species complex), one Fusarium spp, one Fusarium spp. (Fusarium incarnatum-equiseti species complex), and one F. napiforme (Gibberella fujikuroi species complex), isolated from eyes with fungal keratitis were used in this study. Their susceptibility to antibacterial agents: flomoxef, imipenem, gatifloxacin, levofloxacin, moxifloxacin, gentamicin, tobramycin, and Tobracin (R) (contained 3,000 mu g/ml of tobramycin and 25 mu g/ml of benzalkonium chloride (BAK), a biocidal agent: BAK, and antifungal agents: amphotericin B, pimaricin (natamycin), fluconazole, itraconazole, miconazole, voriconazole, and micafungin, was determined by broth microdilution tests. The half-maximal inhibitory concentration (IC50), 100% inhibitory concentration (IC100), and minimum inhibitory concentration (MIC) against the Fusarium isolates were determined. BAK had the highest activity against the Fusarium spp. except for the antifungal agents. Three fluoroquinolones and two aminoglycosides had inhibitory effects against the Fusarium spp. at relatively high concentrations. Tobracin (R) had a higher inhibitory effect against Fusarium spp. than tobramycin alone. Amphotericin B had the highest inhibitory effect against the Fusarium spp, although it had different degrees of activity against each isolate. Our findings showed that fluoroquinolones, aminoglycosides, and BAK had some degree of inhibitory effect against the seven Fusarium isolates, although these agents had considerably lower effect than amphotericin B. However, the inhibitory effects of amphotericin B against the Fusarium spp. varied for the different isolates. Further studies for more effective medications against Fusarium, such as different combinations of antibacterial, biocidal, and antifungal agents are needed.

The incidence of Aspergillus infection has been increasing in the past few years. Also, new Aspergillus fumigatus-related species, namely Aspergillus lentulus, Aspergillus udagawae, and Aspergillus viridinutans, were shown to infect humans. These fungi exhibit marked morphological similarities to A. fumigatus, albeit with different clinical courses and antifungal drug susceptibilities. The present study used liquid chromatography/time-of-flight mass spectrometry to identify the secondary metabolites secreted as virulence factors by these Aspergillus species and compared their antifungal susceptibility. The metabolite profiles varied widely among A. fumigatus, A. lentulus, A. udagawae, and A. viridinutans, producing 27, 13, 8, and 11 substances, respectively. Among the mycotoxins, fumifungin, fumiquinazoline A/B and D, fumitremorgin B, gliotoxin, sphingofungins, pseurotins, and verruculogen were only found in A. fumigatus, whereas auranthine was only found in A. lentulus. The amount of gliotoxin, one of the most abundant mycotoxins in A. fumigatus, was negligible in these related species. In addition, they had decreased susceptibility to antifungal agents such as itraconazole and voriconazole, even though metabolites that were shared in the isolates showing higher minimum inhibitory concentrations than epidemiological cutoff values were not detected. These strikingly different secondary metabolite profiles may lead to the development of more discriminative identification protocols for such closely related Aspergillus species as well as improved treatment outcomes. (C) 2015, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Aspergillus fumigatus is the Aspergillus species most commonly associated with aspergillosis. Of the various presentations of aspergillosis, one of the most frequently observed in cases involving A. fumigatus pulmonary infections is aspergilloma (PA). In such infections one finds a fungus ball composed of fungal hyphae, inflammatory cells, fibrin, mucus, and tissue debris. Chronic necrotizing pulmonary aspergillosis (CNPA), also known as semi-invasive or invasive aspergillosis, is locally invasive and predominantly seen in patients with mild immunodeficiency or with a chronic lung disease. In the present study, with the aid of a next-generation sequencer, we conducted whole genome sequence (WGS) analyses of 17 strains isolated from patients in Japan with PA and CNPA. A total of 99,088 SNPs were identified by mapping the reads to A. fumigatus genome reference strain Af293, and according to genome-wide phylogenetic analysis, there were no correlations between the whole genome sequence typing results and pathologic conditions of patients. Here, we conducted the first multi-genome WGS study to focus on the A. fumigatus strains isolated from patients with PA and CNPA, and comprehensively characterized genetic variations of strains. WGS approach will help in better understanding of molecular mechanisms of aspergillosis cases caused by A. fumigatus.

Cryptococcosis due to a highly virulent fungus, Cryptococcus gattii, emerged as an infectious disease on Vancouver Island in Canada and surrounding areas in 1999, causing deaths among immunocompetent individuals. Previous studies indicated that C. gattii strain R265 isolated from the Canadian outbreak had immune avoidance or immune suppression capabilities. However, protective immunity against C. gattii has not been identified. In this study, we used a gain-of-function approach to investigate the protective immunity against C. gattii infection using a dendritic cell (DC)-based vaccine. Bone marrow-derived dendritic cells (BMDCs) efficiently engulfed acapsular C. gattii (Delta cap60 strain), which resulted in their expression of costimulatory molecules and inflammatory cytokines. This was not observed for BMDCs that were cultured with encapsulated strains. When Delta cap60 strain-pulsed BMDCs were transferred to mice prior to intratracheal R265 infection, significant amelioration of pathology, fungal burden, and the survival rate resulted compared with those in controls. Multinucleated giant cells (MGCs) that engulfed fungal cells were significantly increased in the lungs of immunized mice. Interleukin 17A (IL-17A)-, gamma interferon (IFN-gamma)-, and tumor necrosis factor alpha (TNF-alpha)-producing lymphocytes were significantly increased in the spleens and lungs of immunized mice. The protective effect of this DC vaccine was significantly reduced in IFN-gamma knockout mice. These results demonstrated that an increase in cytokine-producing lymphocytes and the development of MGCs that engulfed fungal cells were associated with the protection against pulmonary infection with highly virulent C. gattii and suggested that IFN-gamma may have been an important mediator for this vaccine-induced protection.

Background: The MALDI (matrix-assisted laser desorption/ionization) Biotyper system for bacterial identification has already been utilized in clinical microbiology laboratories as a successful clinical application of protoemics. However, in cases of Nocardia, mass spectra suitable for MALDI Biotyper identification are often not obtained if such specimens are processed like general bacteria. This problem is related to the insufficiencies in bacterial spectrum databases that preclude accurate specimen identification. Here, we developed a bacterial processing method to improve mass spectra from specimens of the genus Nocardia. In addition, with the new processing method, we constructed a novel in-house bacterial database that combines a commercial database and mass spectra of Nocardia strains from the Department of Clinical Laboratory at Chiba University Hospital (DCLC) and the Medical Mycology Research Center at Chiba University (MMRC). Results: The newly developed method (Nocardia Extraction Method at DCLC [NECLC]) based on ethanol-formic acid extraction (EFAE) improved mass spectra obtained from Nocardia specimens. The Nocardia in-house database at Chiba University Hospital (NDCUH) was then successfully validated. In brief, prior to introduction of the NECLC and NDCUH, 10 of 64 (15.6%) clinical isolates were identified at the species level and 16 isolates (25.0%) could only be identified at the genus level. In contrast, after the introduction, 58 isolates (90.6%) were identified at the species level and 6 isolates (9.4%) were identified at the genus level. Conclusions: The results of this study suggest that MALDI-TOF (time-of-flight) Biotyper system can identify Nocardia accurately in a short time in combination with a simple processing method and an in-house database.

肺ノカルジア症の臨床的な特徴に関する報告は限られている。2003〜2014年に自施設で経験した肺ノカルジア症12例を、後方視的に検討した。平均年齢は69歳、男性10例。経過中に8例の混合感染を認めた。ST合剤を投与した9例中5例は、副作用のため他薬へ変更した。観察期間内に本症による喀血死1例を認め、5例が他病死した。結論として、肺ノカルジア症には混合感染がまれではなく、ST合剤は副作用の頻度が高い。本症の予後については、合併症の影響が無視できない。(著者抄録)

Histoplasmosis, one of the most important mycoses, needs to be diagnosed rapidly and accurately. The main method used to diagnose histoplasmosis is serological detection of antibodies to the Histoplasma capsulatum H and M antigens. Several other protein antigens have been reported in H. capsulatum; however, they have not been used for diagnosis. In this study, we explored novel antigens that were detected during H. capsulatum infection. We obtained a protein mixture from H. capsulatum yeast cells after vigorous mixing in a 0.1% Triton X-100 solution. From the resultant pool, we detected nine spots that reacted with sera from patients with histoplasmosis and identified eight seroactive proteins with mass spectrometry. The seroactive proteins were purified, and their antigenicities were tested with an enzyme-linked immunosorbent assay (ELISA). ELISA revealed that the titer of the patients' sera to N-acetylated α-linked acidic dipeptidase was significantly higher than those of healthy volunteers (P < 0.01). These data indicate that N-acetylated α-linked acidic dipeptidase of H. capsulatum is recognized as a major antigen during histoplasmosis.

Candida parapsilosis complex (CPC) is the third Candida species isolated in blood cultures of patients from our Hospital, following C. albicans and C. tropicalis. From 2006 to 2010, the median annual distribution of CPC was 8 cases/year. Records of 36 patients were reviewed. CPC were 31 (86.1 %) C. parapsilosis; 4 (11.1 %) C. orthopsilosis; and 1 (2.8 %) C. metapsilosis. Clinical characteristics were central venous catheter, 34 (94.4 %); parental nutrition, 25 (70 %); surgery, 27 (57.9 %); prior bacteremia, 20 (51.3 %); malignancy, 18 (50 %). General mortality was 47.2 %. Death was higher in immunosuppressed patients (17 vs. 11; p = 0.003). Three out four (75 %) patients with C. orthopsilosis and 14 out 31 (45.2 %) with C. parapsilosis died (p = 0.558). Thirty-nine individual isolates were tested for susceptibility to seven antifungal drugs, with MICs values showing susceptibility to all of them. Two isolates, one C. orthopsilosis and one C. parapsilosis, had fluconazole MIC = 4 mu g/mL. Differentiation among CPC has implication in caring for patients with invasive candidiasis since there are differences in virulence, pathogenicity and drug susceptibility. A method targeting the topoisomerase II gene based on loop-mediated isothermal amplification (LAMP) was developed. LAMP emerges as a promising tool for the identification of fungal species due to the high sensitivity and specificity. LAMP can be performed at the point-of-care, being no necessary the use of expensive equipment. In our study, the method was successful comparing to the DNA sequencing and proved to be a reliable and fast assay to distinguish the three species of CPC.

The emergence of azole-resistant strains of Aspergillus fumigatus during treatment for aspergillosis occurs by a mutation selection process. Understanding how antifungal resistance mechanisms evolve in the host environment during infection is of great clinical importance and biological interest. Here, we used next-generation sequencing (NGS) to identify mutations that arose during infection by A. fumigatus strains sequentially isolated from two patients, one with invasive pulmonary aspergillosis (IPA) (five isolations) and the other with aspergilloma (three isolations). The serial isolates had identical microsatellite types, but their growth rates and conidia production levels were dissimilar. A whole-genome comparison showed that three of the five isolates from the IPA patient carried a mutation, while 22 mutations, including six nonsynonymous ones, were found among three isolates from the aspergilloma patient. One aspergilloma isolate carried the cyp51A mutation P216L, which is reported to confer azole resistance, and it displayed an MIC indicating resistance to itraconazole. This isolate harbored five other nonsynonymous mutations, some of which were found in the afyap1 and aldA genes. We further identified a large deletion in the aspergilloma isolate in a region containing 11 genes. This finding suggested the possibility that genomic deletions can occur during chronic infection with A. fumigatus. Overall, our results revealed dynamic alterations that occur in the A. fumigatus genome within its host during infection and treatment.

Aspergillus fumigatus is a life-threatening pathogenic fungus, whose conidium is the infectious agent of aspergillosis. To better understand the mechanism underlying the long-term viability of conidia, we characterized a bZip transcription factor, AtfA, with special reference to stress-tolerance in conidia. The atfA deletion mutant conidia showed significant sensitivity to high temperature and oxidative stress. The trehalose content that accumulated in conidia was reduced in the mutant conidia. Transcriptome analysis revealed that AtfA regulated several stress-protection-related genes such as catA, dprA, scf1, and conj at the conidiation stage. The upstream high-osmolarity glycerol pathway was also involved in conferring stress tolerance in conidia because Delta pbsB showed stress sensitivity and reduced trehalose in conidia. However, a mutant lacking the SakA mitogen-activated protein kinase (MAPK) produced normal conidia. We investigated another MAPK, MpkC, in relation with SakA, and the double deletion mutant, Delta sa-kA,mpkC, was defective in conidia stress tolerance. We concluded that MpkC is able to bypass SakA, and the two MAPKs redundantly regulate the conidia-related function of AtfA in A. fumigatus. (C) 2014 Elsevier Inc. All rights reserved.

Background: Pulmonary nocardiosis frequently occurs in immunocompromised hosts and in some immunocompetent hosts with chronic lung disease; however, few reports have described pulmonary nocardiosis with nontuberculous mycobacterial lung infection. Here we report for the first time two cases of pulmonary nocardiosis caused by Nocardia cyriacigeorgica associated with Mycobacterium avium complex (MAC) lung disease caused by M. avium. Case presentation: Case 1 is that of a 72-year-old Japanese man with untreated MAC lung disease, who was diagnosed with rheumatoid arthritis and initiated on methotrexate. After 3 years of methotrexate therapy, the patient remained smear-negative and culture-positive for MAC, but also became smear-positive for Nocardia species. He received trimethoprim/sulfamethoxazole, and his symptoms and lung infiltrates improved. Case 2 is that of an immunocompetent 53-year-old Japanese woman with MAC lung disease, who was treated with a combined therapy of clarithromycin, rifampicin, ethambutol, and levofloxacin. MAC sputum culture was negative after 1 year of combined treatment, which was maintained for 2 years. After four treatment-free years, Nocardia species were occasionally isolated from her sputum, although MAC was rarely isolated from sputum cultures over the same period. In both cases, the Nocardia species were identified as the recently defined N. cyriacigeorgica by 16S ribosomal RNA gene sequencing. Conclusion: We report two cases of pulmonary nocardiosis caused by N. cyriacigeorgica associated with MAC lung disease caused by M. avium and suggest that N. cyriacigeorgica may be a major infective agent associated with MAC lung disease.

The lungs are known to be a major focus of infection by various fungi as they are an important port of entry for the same. Among them, Aspergillus and Cryptotococcus are the most well-known, but many other fungi can cause pulmonary infection. The causative agents of these "rare" pulmonary mycoses include zygomycetes such as Rhizopus spp. along with Cunnnighamella bertholletiae, Schizophyllum commune, and Coccidioides immitis. Although no official data on their epidemiology is present, the prevalence of these diseases is considered to be on the rise. Each disease has its own characteristic pathophysiology, and requires specific diagnostic and therapeutic measures.